Novel application of heparin-binding epidermal growth factor-like growth factor for medical purposes

ABSTRACT

The present invention provides an agent for protecting the liver and/or promoting liver regeneration, which contains a heparin-binding EGF-like growth factor-like growth factor (HB-EGF) or a partial peptide thereof, or a nucleic acid that encodes same, and an agent for the prophylaxis or treatment of liver diseases. The present invention further provides a method for producing a cell for liver protection and/or promoting liver regeneration, and for the prophylaxis/treatment of a liver disease, which includes introducing a nucleic acid that encodes HB-EGF or a partial peptide thereof into a cell collected from an animal.

TECHNICAL FIELD

The present invention relates to a novel pharmaceutical use of aheparin-binding EGF-like growth factor (hereinafter to be abbreviated as“HB-EGF”) or a nucleic acid encoding the same, and more specifically,use for the prophylaxis/treatment of a liver disease.

BACKGROUND ART

Pharmaceutical agents and treatment methods for liver diseases currentlyused in clinical situations merely aim at symptomatic therapy, and thereis no definitive agent or definitive therapy that inhibits liver damage(hepatocyte death) and induces liver regeneration. At present,therefore, the primary course of treatment for acute hepatitis is restand diet. In fact, none of the pharmaceutical agents currently in usehas demonstrated true effectiveness for the disease. In particular,although various pharmaceutical agents and treatment methods have beenemployed for fulminant hepatic failure, the death rate from the diseaseis still close to 70%, proving that they are far from effectivepharmaceutical agents and treatment methods. In Europe and the UnitedStates, liver transplantation is the first choice of treatment.

As stated above, in the actual situation, a definitive drug for liverdiseases (particularly fulminant hepatic failure) does not exist at theclinical level. Under the circumstances, hepatocyte growth factor (HGF)has been confirmed effective at the research level, and is expected mostfor practical application soon. One of the present inventors previouslyreported that HGF strongly inhibits hepatocyte death and simultaneouslyinduces liver regeneration after liver damage in a fulminant hepaticfailure model (WO 98/24467; Kosai, K. et al., Hum. Gene Ther., 92:1293-1301 (1998); Kosai, K. et al., Biochem. Biophys. Res. Commun., 244:683-90 (1998); Kosai, K. et al., Hepatology, 30: 151-9 (1999)), and thedevelopment is ongoing for the clinical application.

HB-EGF is a growth factor belonging to the EGF family, which is alsoexpressed in normal liver. It has biologically unique characteristics inthat it is first synthesized as a membrane-binding type precursor(proHB-EGF), cleaved by a specific metalloproteinase at thejuxtamembrane domain, and the resulting soluble HB-EGF shows a mitogenicaction on many types of cells. In the liver, HB-EGF shows a sharpincrease in expression after hemihepatectomy, and also shows promotedexpression in chronic liver diseases. Based on these facts, HB-EGF hasbeen, like HGF and the like, presumed to be one of the hepatotrophicfactors.

Moreover, it has been reported that the administration of and genetherapy with HB-EGF show a therapeutic effect on some of the organdisorders. For example, it has been suggested that administration ofrecombinant HB-EGF (including topical production by gene therapy) todamaged epithelial cells of patients with interstitial cystitis (IC) canblock the effect of an antiproliferative factor (APF) causing epithelialabnormality in IC, and alleviate or remove chronic damage in the bladderepithelium (WO 99/54706). Furthermore, an backward injection of anadenoviral vector containing HB-EGF gene to a diabetic mouse from thepancreatic duct affords growth of pancreatic β cells, differentiation ofpancreatic duct cells into insulin-producing cells and improvement inglucose tolerance, suggesting that HB-EGF shows a pancreas regenerationand⋅diabetes treating effect (Kozawa, J. et al., Pancreas, 31: 32-42(2005)).

However, no report has so far documented that HB-EGF actually shows atherapeutic effect on liver diseases, namely, exogenous HB-EGFsuppresses liver damage and/or induces liver regeneration. Moreover,although HB-EGF is, like HGF and insulin-like growth factor (IGF), knownas a cardiohypertrophic growth factor, a gene therapy using HB-EGF didnot show any therapeutic effect unlike use of HGF or IGF, in amyocardial infarction treatment test using rabbit conducted by thepresent inventors. Conversely, it rather aggravated remodeling afterinfarction (Ushikoshi, H. et al., Lab. Invest., 85: 862-73 (2005)).Thus, it seems that HB-EGF does not necessarily act therapeutically onall organ disorders.

As described above, it is completely unknown whether or not HB-EGFactually shows a therapeutic effect on liver diseases, not to mentionthe effectiveness of its gene therapy which provides no guarantee atall.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a noveland more useful prophylactic or therapeutic drug for a liver disease(particularly fulminant hepatic failure) by identifying a substance thatshows a stronger liver damage suppressing and liver regenerationinducing action than HGF does.

The present inventors have introduced and expressed an adenoviral vectorcontaining a DNA encoding HB-EGF in an animal fulminant hepatic failuremodel and found that HB-EGF remarkably suppresses liver damage andhepatocyte apoptosis and induces liver regeneration. More surprisingly,the treatment effects of HB-EGF have been found to be stronger thanthose of HGF, the currently-known most promising therapeutic drug forliver diseases. The inventors have conducted further investigationsbased on these findings, and completed the present invention.

Accordingly, the present invention provides an agent for protecting theliver and/or promoting liver regeneration, which comprises a nucleicacid encoding HB-EGF or a partial peptide thereof. When the nucleic acidis introduced into target cells by way of, for example, a viral vectordescribed below or the like, into which the nucleic acid has beeninserted, it is intracellularly expressed to produce and release HB-EGFor a partial peptide thereof, and suppresses liver damage (hepatocytedeath) as well as induces liver regeneration.

Therefore, the present invention also provides an agent for protectingthe liver and/or promoting liver regeneration, which comprises HB-EGF ora partial peptide thereof. The protein (or peptide) can be produced inlarge amounts using the gene recombination technique as described below,and used.

Furthermore, the present invention provides a method of producing cellsfor protecting the liver and/or promoting liver regeneration, whichcomprises introducing a nucleic acid encoding HB-EGF or a partialpeptide thereof into cells collected from an animal. By putting back thecells collected from an individual, into which the nucleic acid has beenintroduced and whose expression has been confirmed, to the individual orimplanting the cells into a different individual, liver damage(hepatocyte death) is suppressed and liver regeneration is induced inthe animal individual.

Since HB-EGF shows a strong liver damage suppressive, apoptosissuppressive and liver regeneration inducing action, it provides aprophylactic or therapeutic effect on various diseases accompanyingliver damage or hepatocyte death by administration of HB-EGF or anucleic acid encoding HB-EGF.

Specific embodiments of the present invention and other advantages andthe like of the present invention are described in more detail in “BestMode for Embodiment of the Invention” below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show that the target gene can be efficiently introducedinto the liver and hepatocyte by injection of an adenoviral vector fromthe tail vein. FIG. 1A shows that X-gal staining positive cells areobserved throughout the liver tissues after Ad.LacZ introduction. FIG.1B shows that HB-EGF expression is observed at the edge of the cellularmembrane by introducing Ad.HB-EGF.

FIGS. 2A, 2B and 2C show an influence of the administration ofAd.HB-EGF, Ad.HGF or Ad.dE1.3 on the blood concentration of liver enzymeof fulminant hepatic failure model mice. FIG. 2A shows the schedule ofthe experiment. FIG. 2B shows the serum ALT value and FIG. 2C shows theserum AST value (white bar: 24 hr after antibody administration, blackbar: 36 hr after antibody administration).

FIG. 3 shows an influence of the administration of Ad.HB-EGF (middle),Ad.HGF (bottom) or Ad.dE1.3 (top) on liver tissue apoptosis in fulminanthepatic failure model mice. The left side and right side images aremicroscopic images of liver tissues of each administration group mouse24 hr after administration and 36 hr after administration, respectively.

FIGS. 4A and 4B show the results of TUNEL staining of the livers offulminant hepatic failure model mice administered with Ad.HB-EGF, Ad.HGFor Ad.dE1.3. FIG. 4A shows, from the left, microscopic images of theAd.dE1.3-, Ad.HB-EGF-, or Ad.HGF-administration group mice (top: 24 hrafter antibody administration, bottom: 36 hr after antibodyadministration). FIG. 4B is a graph showing quantified TUNEL-positivecells (white bar: 24 hr after antibody administration, black bar: 36 hrafter antibody administration).

FIGS. 5A and 5B show an influence of the administration of Ad.HB-EGF,Ad.HGF or Ad.dE1.3 on liver regeneration in fulminant hepatic failuremodel mice. FIG. 5A shows, from the left, Ki-67-stained images of thelivers of the Ad.dE1.3-, Ad.HB-EGF- or Ad.HGF-administration group mice(top: 24 hr after antibody administration, bottom: 36 hr after antibodyadministration). FIG. 5B is a graph showing quantified Ki-67-positivecells (white bar: 24 hr after antibody administration, black bar: 36 hrafter antibody administration).

BEST MODE FOR EMBODIMENT OF THE INVENTION

With regard to HB-EGF used for the present invention, derivation thereofis not particularly limited, as long as it is a protein containing thesame or substantially the same amino acid sequence as an amino acidsequence shown by SEQ ID NO:2, and it can be a protein derived from acell [for example, hepatocyte, splenocyte, nerve cell, glial cell,pancreatic β cell, bone marrow cell, mesangial cell, Langerhans cell,epidermal cell, epithelial cell, goblet cell, endothelial cell, smoothmuscle cell, skeletal muscle cell, fibroblast, fiber cell, adipocyte,immune cell (e.g., macrophage, T cell, B cell, natural killer cell, mastcell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte,synovial cell, chondrocyte, osteocyte, osteoblast, osteoclast, mammarycell or interstitial cell, or precursor cell, stem cell, established orcancer cell thereof, and the like] of human or other warm-blooded animal(e.g., bovine, swine, mouse, rat, hamster, monkey, horse, sheep, goat,rabbit, guinea pig, chicken and the like) or any tissue or organ inwhich these cells are present [for example, brain, any portion of brain(e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus,thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum),spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad,thyroid gland, gall bladder, bone marrow, adrenal gland, skin, muscle(skeletal muscle, smooth muscle), lung, gastrointestinal tract (e.g.,large intestine, small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, urinary duct, prostate, orchis,ovary, placenta, uterus, bone, joint, adipose tissue (e.g., brownadipose tissue, white adipose tissue) and the like], or may be asynthetic protein synthesized chemically or biochemically using acell-free protein synthesis system as described below. Alternatively,this protein may be a recombinant protein produced from a transformantintroduced with a nucleotide having the base sequence encoding theabove-described amino acid sequence.

Substantially the same amino acid sequence as the amino acid sequenceshown by SEQ ID NO:2 refers to an amino acid sequence having a homologyof about 70% or more, preferably about 80% or more, more preferablyabout 90% or more, and particularly preferably about 95% or more, to theamino acid sequence shown by SEQ ID NO:2, wherein the proteinscontaining the amino acid sequence have substantially the identicalactivity to the protein containing the amino acid sequence shown by SEQID NO:2. “Homology” herein means a proportion (%) of the same amino acidresidue and analogous amino acid residue to the whole amino acidresidues overlapped in the optimal alignment (preferably, the algorithmis such that a gap can be introduced into one or both of the sequencesfor an optimal alignment) where two amino acid sequences are alignedusing a mathematic algorithm known in the technical field. The“analogous amino acid” means amino acids having similar physiochemicalproperties, and, for example, the amino acids are classified into groupssuch as an aromatic amino acid (Phe, Trp, Tyr), an aliphatic amino acid(Ala, Leu, Ile, Val), a polar amino acid (Gln, Asn), a basic amino acid(Lys, Arg, His), an acidic amino acid (Glu, Asp), an amino acid having ahydroxy group (Ser, Thr) and an amino acid having a small side-chain(Gly, Ala, Ser, Thr, Met). Substitution by such analogous amino acids isexpected not to change the phenotype of proteins (i.e., conservativeamino acid substitution). Specific examples of the conservative aminoacid substitution are known in this technical field and described invarious literatures (e.g., see Bowie et al., Science, 247: 1306-1310(1990)).

The homology of the amino acid sequence in the present specification canbe calculated using homology calculation algorithm NCBI BLAST (NationalCenter for Biotechnology Information Basic Local Alignment Search Tool)under the following conditions (expectancy=10; allowing gap;matrix=BLOSUM62; filtering=OFF). Other algorithms to determine ahomology of amino acid sequence include, for example, the algorithm asdescribed in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877(1993) [the algorithm is incorporated into NBLAST and XBLAST programs(version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402(1997))], the algorithm as described in Needleman et al., J. Mol. Biol.,48: 444-453 (1970) [the algorithm is incorporated into a GAP program ina GCG software package], the algorithm as described in Myers and Miller,CABIOS, 4: 11-17 (1988) [the algorithm is incorporated into an ALIGNprogram (version 2.0) which is a part of a CGC sequence alignmentsoftware package], the algorithm as described in Pearson et al., Proc.Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm isincorporated into a FASTA program in a GCG software package], and thelike, which can be preferably used in a similar manner.

More preferable, substantially the same amino acid sequence as the aminoacid sequence shown by SEQ ID NO:2 is an amino acid sequence having ahomology of about 60% or more, preferably about 70% or more, morepreferably about 80% or more, and particularly preferably about 90% ormore, to the amino acid sequence shown by SEQ ID NO:2.

As substantially the identical activity, for example, liver protection(inhibition of liver damage) action, apoptosis (hepatocyte death)inhibitory action, liver regeneration (differentiation and/or divisionof hepatocyte)-inducing action and the like can be mentioned.“Substantially the identical” means that these activities arequalitatively (e.g., physiologically or pharmacologically) the same.Therefore, the activities such as liver protection action, apoptosisinhibitory action and liver regeneration-inducing action are preferablyequivalent (e.g., about 0.5- to about 2-fold), but quantitative factorssuch as the extent of activity and protein molecular weight may bedifferent.

Measurement of the activities such as liver protection action, apoptosisinhibitory action and liver regeneration-inducing action can beperformed according to a method known per se, which can include, but isnot limited to, for example, methods as described in the below-mentionedExample such as histophathological observation (e.g., hematoxylin-eosin(H-E) stain), TUNEL assay, and immunohistochemistry using aproliferation marker such as Ki67 antigen as an index, of a liver tissuesection, and the like, respectively.

HB-EGF of the present invention includes, for example, a proteincontaining (1) an amino acid sequence shown in SEQ ID NO:2 wherein oneor more [preferably, about 1-50, preferably about 1-30, more preferablyabout 1-10, particularly preferably 1 to several (2, 3, 4 or 5)] aminoacids have been deleted, (2) an amino acid sequence shown in SEQ ID NO:2wherein one or more [preferably, about 1-50, preferably about 1-30, morepreferably about 1-10, particularly preferably 1 to several (2, 3, 4 or5)] amino acids have been added, (3) an amino acid sequence shown in SEQID NO:2 wherein one or more [preferably, about 1-50, preferably about1-30, more preferably about 1-10, particularly preferably 1 to several(2, 3, 4 or 5)] amino acids have been inserted, (4) an amino acidsequence shown in SEQ ID NO:2 wherein one or more [preferably, about1-50, preferably about 1-30, more preferably about 1-10, particularlypreferably 1 to several (2, 3, 4 or 5)] amino acids have beensubstituted by other amino acids, or (5) an amino acid sequence whereinthe above-mentioned sequences have been combined, and the like.

When an amino acid sequence is inserted, deleted or substituted asdescribed above, the position of the insertion, deletion or substitutionis not subject to limitation, as long as the protein retains itsactivity. As the position affecting the activity of HB-EGF can include,for example, an EGF-like domain (a region shown by amino acid numbers105 to 148 in the amino acid sequence shown by SEQ ID NO:2), which isassociated with binding with EGFR, a HB-EGF receptor, proteaserecognition and cleavage sites in shedding, and the like can bementioned and, therefore, as the position of the insertion, deletion andsubstitution, for example, prosequence, C-terminal intracellular domainand the like can be mentioned. Alternatively, since it has been reportedthat heparin-binding domain (a region shown by amino acid numbers 93 to105 in the amino acid sequence shown by SEQ ID NO:2) regulates bindingactivity of HB-EGF and EGFR negatively (J. Biol. Chem., 279(45):47335-43 (2004)), the deletion, insertion and substitution in the domaincan also maintain or enhance the activity of HB-EGF.

HB-EGF of the present invention is preferably human HB-EGF (GenBankAccession Number: NP_001936) having the amino acid sequence shown in SEQID NO:2, or an ortholog thereof in an other warm-blooded animal [forexample, orthologs in mouse, rat, bovine, swine, Chinese hamster andchicken registered in Genbank under Accession Number NP_034545,NP_037077, XP_601210, NP_999464, AAD52998 and NP_990180 (having about81%, about 82%, about 73%, about 91%, about 86% and about 73%, homologyto human HB-EGF, respectively), respectively, and the like].

For the proteins and peptides mentioned herein, the left end is the Nterminal (amino terminal) and the right end is the C terminal (carboxylterminal) in accordance with the conventional peptide marking. RegardingHB-EGF of the present invention, including a protein comprising theamino acid sequence shown by SEQ ID NO:2, the C terminal may be any of acarboxyl group (—COOH), a carboxylate (—COO⁻), an amide (—CONH₂), and anester (—COOR).

Here, as R in the ester, a C₁₋₆ alkyl group such as methyl, ethyl,n-propyl, isopropyl, and n-butyl; a C₃₋₈ cycloalkyl group such ascyclopentyl and cyclohexyl; a C₆₋₁₂ aryl group such as phenyl andα-naphthyl; a phenyl-C₁₋₂ alkyl group such as benzyl and phenethyl; aC₇₋₁₄ aralkyl group such as an α-naphthyl-C₁₋₂ alkyl group such asα-naphthylmethyl; a pivaloyloxymethyl group; and the like are used.

When HB-EGF of the present invention has a carboxyl group (or acarboxylate) at a position other than the C terminal, a protein whereinthe carboxyl group is amidated or esterified is also included in theprotein of the present invention. In this case, as the ester, theabove-described ester at the C terminal, and the like, for example, areused.

Furthermore, HB-EGF of the present invention also includes a proteinwherein the amino group of the N terminal amino acid residue isprotected by a protecting group (e.g., C₁₋₆ acyl groups such as C₁₋₆alkanoyl groups such as formyl group and acetyl group; and the like), aprotein wherein the N terminal glutamine residue, which is produced uponcleavage in vivo, has been converted to pyroglutamic acid, a proteinwherein a substituent (e.g., —OH, —SH, amino group, imidazole group,indole group, guanidino group, and the like) on a side chain of an aminoacid in the molecule is protected by an appropriate protecting group(e.g., C₁₋₆ acyl groups such as C₁₋₆ alkanoyl groups such as formylgroup and acetyl group; and the like), a conjugated protein such as whatis called a glycoprotein having a sugar chain bound thereto, and thelike.

A HB-EGF partial peptide may be any peptide having the above-describedHB-EGF partial amino acid sequence, and having substantially the samequality of activity as HB-EGF. Here, “substantially the same quality ofactivity” has the same definition as above. A measurement of“substantially the same quality of activity” can be conducted in thesame manner as in the case of HB-EGF.

Specifically, as HB-EGF partial peptide, for example, in the amino acidsequence shown by SEQ ID NO:2, a partial amino acid sequence containingthe EGF-like domain (mentioned above), which is associated with thebinding with the receptor EGFR, a partial amino acid sequence containinga soluble HB-EGF (consisting of an amino acid sequence shown by aminoacid numbers 63 to 148 in the amino acid sequence shown by SEQ ID NO:2),a partial amino acid sequence further having an intracellular domain(and a juxtamembrane/transmembrane domain) (consisting of an amino acidsequence shown by amino acid numbers 63 to 208 in the amino acidsequence shown by SEQ ID NO:2), and the like can be used.

As HB-EGF partial peptide, preferably a peptide having not less than 40amino acids, more preferably not less than 80 amino acids, still morepreferably not less than 140 amino acids, and the like can be used.

For HB-EGF partial peptide, the C terminal may be any of a carboxylgroup (—COOH), a carboxylate (—COO⁻), an amide (—CONH₂), and an ester(—COOR). Here, as R in the ester, the same as those mentioned for HB-EGFabove can be mentioned. When HB-EGF partial peptide has a carboxyl group(or a carboxylate) at a position other than the C terminal, a partialpeptide wherein the carboxyl group is amidated or esterified is alsoincluded in HB-EGF partial peptide. In this case, as the ester, thatsimilar to the ester at the C terminal, and the like, for example, areused.

Furthermore, HB-EGF partial peptide also includes a protein wherein theamino group of the N terminal amino residue is protected by a protectinggroup, a protein wherein glutamic residue at the N terminal in vivo hasbeen converted to pyroglutamic acid, a protein wherein a substituent ona side chain of an amino acid in the molecule is protected by anappropriate protecting group, a conjugated peptide such as what iscalled a glycopeptide having a sugar chain bound thereto, and the like,as with the above-described HB-EGF.

As the salt of HB-EGF or a partial peptide thereof, physiologicallyacceptable salts with acid (e.g., inorganic acid, organic acid) or base(e.g., alkali metal) can be mentioned, and physiologically acceptableacid addition salts are preferred. Useful salts include, for example,salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid,hydrobromic acid, sulfuric acid) or salts with organic acids (e.g.,acetic acid, formic acid, propionic acid, fumaric acid, maleic acid,succinic acid, tartaric acid, citric acid, malic acid, oxalic acid,benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

HB-EGF or a salt thereof can be produced by a protein purificationmethod known per se from the aforementioned cells or tissues of thewarm-blooded animals. Specifically, HB-EGF or the salt thereof can beprepared by homogenizing the tissue or cell of the warm-blooded animal,then removing debris of the cell by low-speed centrifugation,precipitating a cellular membrane-containing fraction by high-speedcentrifugation of a supernatant (as necessary, purifying the cellularmembrane fraction by density gradient centrifugation and the like), andsubjecting the fraction to a chromatography such as reversed-phasechromatography, ion exchange chromatography and affinity chromatography.When the soluble HB-EGF is prepared as HB-EGF partial peptide, it can beobtained by culturing the tissue or cell of the warm-blooded animal in asuitable medium, followed by subjecting a supernatant obtained byremoving the cell by a filtration or centrifugation and the like to achromatography such as reversed-phase chromatography, ion exchangechromatography and affinity chromatography.

HB-EGF or a partial peptide thereof or a salt thereof (hereinafter alsocomprehensively referred to as “HB-EGFs”) can also be produced accordingto a publicly known method of peptide synthesis.

The method of peptide synthesis may be any of, for example, a solidphase synthesis process and a liquid phase synthesis process. A desiredprotein can be produced by condensing a partial peptide or amino acidcapable of constituting HB-EGF with the remaining portion, and removingany protecting group the resultant product may have.

Here, the condensation and the protecting group removal are conducted inaccordance with methods known per se, for example, the methods indicatedin (i) to (v) below:

-   (i) M. Bodanszky and M. A. Ondetti: Peptide Synthesis, Interscience    Publishers, New York (1966)-   (ii) Schroeder and Luebke: The Peptide, Academic Press, New York    (1965)-   (iii) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics    and experiments of peptide synthesis), published by Maruzen Co.    (1975);-   (iv) Haruaki Yajima and Shunpei Sakakibara: Seikagaku Jikken Koza    (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of    Proteins) IV, 205 (1977)-   (v) Yoshiaki Kiso and Akira Otaka: Zoku Iyakuhin no Kaihatsu, Vol.    14, Peptide Synthesis, published by Hirokawa Shoten (1991).

The thus-obtained protein (peptide) can be purified and isolated by apublicly known method of purification. Here, as examples of the methodof purification, solvent extraction, distillation, columnchromatography, liquid chromatography, recrystallization, a combinationthereof, and the like can be mentioned.

When the protein (peptide) obtained by the above-described method is afree form, it can be converted to an appropriate salt by a publiclyknown method or a method based thereon; conversely, when the protein(peptide) is obtained in the form of a salt, the salt can be convertedto a free form or another salt by a publicly known method or a methodbased thereon.

For the synthesis of HB-EGF, an ordinary commercially available resinfor protein synthesis can be used. As examples of such resins,chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin,aminomethyl resin, 4-benzyloxybenzyl alcohol resin,4-methylbenzhydrylamine resin, PAM resin,4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin and the like canbe mentioned. Using such a resin, an amino acid having an appropriatelyprotected α-amino group and side chain functional group is condensed onthe resin in accordance with the sequence of the desired protein(peptide) according to one of various methods of condensation known perse. At the end of the reaction, the protein or the like is cleaved fromthe resin and at the same time various protecting groups are removed,and a reaction to form an intramolecular disulfide bond is carried outin a highly diluted solution to obtain the desired protein (peptide) oran amide thereof.

For the above-described condensation of protected amino acids, variousactivation reagents which can be used for protein synthesis can be used,and a carbodiimide is preferably used. As the carbodiimide, DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and the like are used.For the activation using these carbodiimides, the protected amino acid,along with a racemation-suppressing additive (e.g., HOBt, HOOBt), may beadded directly to the resin, or the protected amino acid may beactivated in advance as a symmetric acid anhydride or HOBt ester orHOOBt ester and then added to the resin.

Solvents used for the activation of protected amino acids andcondensation thereof with a resin can be appropriately selected fromamong solvents that are known to be usable for protein condensationreactions. As examples of useful solvents, acid amides such asN,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidone;halogenated hydrocarbons such as methylene chloride and chloroform;alcohols such as trifluoroethanol; sulfoxides such as dimethylsulfoxide; amines such as pyridine; ethers such as dioxane andtetrahydrofuran; nitriles such as acetonitrile and propionitrile; esterssuch as methyl acetate and ethyl acetate; suitable mixtures thereof; andthe like can be mentioned. Reaction temperature is appropriatelyselected from the range that is known to be usable for protein bindingreactions, and is normally selected from the range of about −20° C. toabout 50° C. An activated amino acid derivative is normally used from1.5 to 4 times in excess. When a test using the ninhydrin reactionreveals that the condensation is insufficient, sufficient condensationcan be conducted by repeating the condensation reaction withoutelimination of protecting groups. If the condensation is insufficienteven though the reaction is repeated, unreacted amino acids may beacetylated using acetic anhydride or acetylimidazole.

A protecting method and a protecting group for a functional group thatshould not be involved in the reaction of raw materials, a method ofremoving the protecting group, a method of activating a functional groupinvolved in the reaction, and the like can be appropriately selectedfrom among publicly known groups or publicly known means.

As examples of the protecting group for an amino group of the startingmaterial, Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, Fmoc, and the like can be used.

A carboxyl group can be protected, for example, by alkyl esterification(e.g., linear, branched or cyclic alkyl esterification with methyl,ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, and the like), aralkylesterification (e.g., benzyl esterification, 4-nitrobenzylesterification, 4-methoxybenzyl esterification, 4-chlorobenzylesterification, benzhydryl esterification), phenacyl esterification,benzyloxycarbonyl hydrazidation, tertiary butoxycarbonyl hydrazidation,trityl hydrazidation, and the like.

The hydroxyl group of serine can be protected by, for example,esterification or etherification. As examples of a group suitable forthis esterification, lower alkanoyl groups such as an acetyl group,aroyl groups such as a benzoyl group, and groups derived from carbonicacid such as a benzyloxycarbonyl group and an ethoxycarbonyl group, andthe like are used. As examples of a group suitable for etherification, abenzyl group, a tetrahydropyranyl group, a t-butyl group, and the likecan be mentioned.

As examples of the protecting group for the phenolic hydroxyl group oftyrosine, Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, tertiary butyl, and thelike can be used.

As examples of the protecting group for the imidazole of histidine, Tos,4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, Fmoc, and the like are used.

As examples of the method of removing (eliminating) a protecting group,catalytic reduction in a hydrogen stream in the presence of a catalystsuch as Pd-black or Pd-carbon; acid treatment by means of anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethane-sulfonic acid,trifluoroacetic acid, or a mixture solution thereof; base treatment bymeans of diisopropylethylamine, triethylamine, piperidine, piperazine orthe like; and reduction with sodium in liquid ammonia, and the like areused. The elimination reaction by the above-described acid treatment isgenerally carried out at a temperature of about −20° C. to about 40° C.;the acid treatment is efficiently conducted by adding a cationscavenger, for example, anisole, phenol, thioanisole, m-cresol,p-cresol, dimethylsulfide, 1,4-butanedithiol and 1,2-ethanedithiol.Also, a 2,4-dinitrophenyl group used as a protecting group of theimidazole of histidine is removed by thiophenol treatment; a formylgroup used as a protecting group of the indole of tryptophan is removedby acid treatment in the presence of 1,2-ethanedithiol,1,4-butanedithiol, or the like, as well as by alkali treatment with adilute sodium hydroxide solution, dilute ammonia, or the like.

As examples of those obtained by activation of the carboxyl group in thestarting material, a corresponding acid anhydride, an azide, anactivated ester [an ester with an alcohol (e.g., pentachlorophenol,2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol,p-nitrophenol, HONB, N-hydroxysuccimide, N-hydroxyphthalimide, or HOBt)]and the like are used. As examples of those obtained by activation ofthe amino group in the starting material, a corresponding phosphoricamide is used.

In another method of preparing an amide of a protein (peptide), forexample, the α-carboxyl group of the carboxy terminal amino acid isfirst amidated and hence protected, and a peptide chain is elongated toa desired chain length toward the amino group side, thereafter a protein(peptide) having the protecting group for the N terminal α-amino groupof the peptide chain only removed and a protein (peptide) having theprotecting group for the C terminal carboxyl group only removed areprepared, and these proteins or the like are condensed in a mixedsolvent described above. For details about the condensation reaction,the same as above applies. After the protected protein (protectedpeptide) obtained by the condensation is purified, all protecting groupscan be removed by the above-described method to yield a desired crudeprotein (crude peptide). By purifying this crude protein (crude peptide)using various publicly known means of purification, and freeze-dryingthe main fraction, a desired amide of the protein (peptide) can beprepared.

For esters of the protein (peptide), a desired ester of the protein orthe like can be prepared by, for example, condensing the α-carboxylgroup of the carboxy terminal amino acid with a desired alcohol to yieldan amino acid ester, and then treating the ester in the same manner aswith an amide of the above-mentioned protein (peptide).

HB-EGF partial peptide or a salt thereof can also be produced bycleaving HB-EGF or a salt thereof with an appropriate peptidase (e.g.,ADAM9, ADAM12, etc.)

Furthermore, HB-EGF can also be produced by cultivating a transformantcomprising nucleic acid encoding HB-EGF or a partial peptide thereof,and separating and purifying HB-EGF from the culture obtained. Thenucleic acid encoding HB-EGF or a partial peptide thereof can be DNA orRNA, or DNA/RNA chimera. Preferably DNA can be mentioned. Also, thenucleic acid can be double stranded or single stranded. When it isdouble stranded, it can be double stranded DNA, double stranded RNA orDNA:RNA hybrid. When it is single stranded, it can be sense strand(i.e., coding strand) or antisense strand (i.e., non-coding strand).

As the DNA encoding HB-EGF or a partial peptide thereof, genomic DNA,cDNA (cRNA) derived from a cell [for example, hepatocyte, splenocyte,nerve cell, glial cell, pancreatic cell, bone marrow cell, mesangialcell, Langerhans cell, epidermal cell, epithelial cell, goblet cell,endothelial cell, smooth muscle cell, fibroblast, fiber cell, musclecell, adipocyte, immune cell (e.g., macrophage, T cell, B cell, naturalkiller cell, mast cell, neutrophil, basophil, eosinophil, monocyte),megakaryocyte, synovial cell, chondrocyte, osteocyte, osteoblast,osteoclast, mammary cell or interstitial cell, or precursor cell, stemcell, established or cancer cell thereof, and the like] of human orother warm-blooded animal (e.g., monkey, bovine, horse, swine, sheep,goat, rabbit, mouse, rat, guinea pig, hamster, chicken and the like) orany tissue or organ in which these cells are present [for example,olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus,thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum),spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad,thyroid gland, gall bladder, bone marrow, adrenal gland, skin, muscle,lung, gastrointestinal tract (e.g., large intestine, small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, prostate, orchis, ovary, placenta, uterus, bone, joint, adiposetissue (e.g., brown adipose tissue, white adipose tissue), skeletalmuscle and the like], synthetic DNA (RNA) and the like can be mentioned.The genomic DNA and cDNA encoding HB-EGF or a partial peptide thereofcan be directly amplified by Polymerase Chain Reaction (hereinafter tobe abbreviated as “PCR method”) and Reverse Transcriptase-PCR(hereinafter to be abbreviated as “RT-PCR method”) using a genomic DNAfraction and total RNA or mRNA fraction prepared from theabove-mentioned cell/tissue as templates, respectively. Alternatively,the genomic DNA and cDNA encoding HB-EGF or a partial peptide thereofcan be cloned by colony or plaque hybridization method, or PCR methodand the like, from a genome DNA library and cDNA library prepared byinserting a fragment of genomic DNA and total RNA or mRNA prepared fromthe above-mentioned cell/tissue into a suitable vector. The vector usedfor the library may be any of a bacteriophage, a plasmid, a cosmid, aphagemid and the like.

As examples of the nucleic acids encoding HB-EGF, nucleic acidscontaining a base sequence represented by SEQ ID NO:1 (with the provisothat when the nucleic acid is RNA, “t” in the base sequence is to beread as “u”), nucleic acids containing a base sequence capable ofhybridizing to a complementary strand sequence of the base sequencerepresented by SEQ ID NO:1 under stringent conditions, and encoding aprotein having substantially the identical activity to HB-EGF mentionedabove [e.g.: binding activity to EGFR, liver protection (inhibition ofliver damage) action, apoptosis (hepatocyte death) inhibitory action,liver regeneration (differentiation and/or division ofhepatocyte)-inducing action and the like], and the like can bementioned.

The nucleic acids capable of hybridizing to a complementary strandsequence of the base sequence represented by SEQ ID NO:1 under stringentconditions used include, for example, nucleic acids containing a basesequence having not less than about 60%, preferably not less than about70%, more preferably not less than about 80%, and particularlypreferably not less than about 90%, homology to the base sequencerepresented by SEQ ID NO:1, and the like.

The homology of the base sequence in the present specification can becalculated using homology calculation algorithm NCBI BLAST (NationalCenter for Biotechnology Information Basic Local Alignment Search Tool)under the following conditions (expectancy=10; allowing gap;filtering=ON; match score=1; mismatch score=−3). Examples of otheralgorithms to determine a homology of base sequence preferably includethe above-mentioned homology calculation algorithms of amino acidsequence in a similar manner.

Hybridization can be conducted according to a method known per se or amethod based thereon, for example, a method described in MolecularCloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press,1989) and the like. When a commercially available library is used,hybridization can be conducted according to the method described in theinstruction manual attached thereto. Hybridization can preferably beconducted under highly stringent conditions.

The stringent conditions are exemplified by reaction conditionscharacterized in that (1) a low ionic strength and a high temperature,for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1%dodecyl sodiumsulfate at 50° C., is used for washing, and (2) adenaturing agent such as formamide, for example, 50% (v/v) formamidealong with a 50 mM sodium phosphate buffer (pH 6.5) containing 0.1%bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/750 mM sodiumchloride and 75 mM sodium citrate is used at 42° C. Alternatively, thestringent condition can be a condition in which 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhart's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate are used at 42° C.,and a washing is performed with 0.2×SSC and 50% formaldehyde at 55° C.,followed by a high-stringent washing comprised of EDTA-containing0.1×SSC at 55° C. Those of ordinary skill in the art can easily achievea desired stringency by appropriately adjusting temperature athybridization reaction and/or washing, ion strength of buffer, and thelike based on factors such as probe length.

The nucleic acid encoding HB-EGF is, preferably, a nucleic acidcontaining a base sequence encoding human HB-EGF shown by a basesequence represented by SEQ ID NO:1 (GenBank Accession Number:NM_001945), or an ortholog thereof in an other warm-blooded animal [forexample, orthologs in mouse, rat, bovine, swine, Chinese hamster andchicken registered in GenBank under Accession Number NM_010415,NM_012945, XM_601210, NM_214299, AF069753 and NM_204849 (having about83%, about 83%, about 74%, about 88%, about 74% and about 63%, homologyto human HB-EGF cDNA, respectively), respectively, and the like].

The nucleic acid encoding HB-EGF partial peptide of the presentinvention may be any one comprising the base sequence encoding the sameor substantially the same amino acid sequence as a portion of the aminoacid sequence shown by SEQ ID NO:2. The DNA may be any of genomic DNA,cDNA derived from the above-described cell or tissue, a cDNA (cRNA)derived from the above-described cell or tissue, and synthetic DNA(RNA). The vector used for the library may be any of a bacteriophage, aplasmid, a cosmid, a phagemid and the like. The DNA can also beamplified directly by the RT-PCR method using an mRNA fraction preparedfrom the above-described cell or tissue.

Specifically, as the nucleic acid encoding HB-EGF partial peptide, forexample, (1) a nucleic acid having a partial base sequence of the basesequence represented by SEQ ID NO:1, (2) a nucleic acid containing abase sequence hybridizing to a nucleic acid having the base sequencerepresented by SEQ ID NO:1 under stringent conditions, and encoding apeptide having the substantially the identical activity to HB-EGF [e.g.:binding activity to EGFR, liver protection (inhibition of liver damage)action, apoptosis (hepatocyte death) inhibitory action, liverregeneration (differentiation and/or division of hepatocyte)-inducingaction and the like] mentioned above, or the like is used.

As examples of the DNA capable of hybridizing to the base sequence shownby SEQ ID NO:1 under stringent conditions, a nucleic acid comprising abase sequence showing a homology of about 60% or more, preferably about70% or more, more preferably about 80% or more, and particularlypreferably about 85% or more, to the base sequence, and the like areused.

The DNA encoding HB-EGF or a partial peptide thereof can be cloned byamplifying it by the PCR method using a synthetic DNA primer comprisinga portion of the base sequence encoding HB-EGF or a partial peptidethereof, or by hybridizing DNA incorporated in an appropriate expressionvector to a labeled DNA fragment or synthetic DNA encoding a portion orthe entire region of HB-EGF protein. Hybridization can be conductedaccording to, for example, a method described in Molecular Cloning, 2ndedition (ibidem) and the like. When a commercially available library isused, hybridization can be conducted according to the method describedin the instruction manual attached to the library.

The base sequence of DNA can be converted according to a method knownper se, such as the ODA-LA PCR method, the Gapped duplex method, theKunkel method and the like, or a method based thereon, using a publiclyknown kit, for example, Mutan™-super Express Km (Takara Shuzo Co.,Ltd.), Mutan™-K (Takara Shuzo Co., Ltd.) and the like.

The cloned DNA can be used as is, or after digestion with a restrictionendonuclease or addition of a linker as desired, depending on thepurpose of its use. The DNA may have the translation initiation codonATG at the 5′ end thereof, and the translation stop codon TAA, TGA orTAG at the 3′ end thereof. These translation initiation codons andtranslation stop codons can be added using an appropriate synthetic DNAadapter.

An expression vector containing DNA encoding HB-EGF or a partial peptidethereof can be produced by, for example, cutting out a desired DNAfragment from the DNA encoding HB-EGF, and joining the DNA fragmentdownstream of a promoter in an appropriate expression vector.

Useful expression vectors include plasmids derived from Escherichia coli(e.g., pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillussubtilis (e.g., pUB110, pTP5, pC194); plasmids derived from yeast (e.g.,pSH19, pSH15); insect cell expression plasmids (e.g., pFast-Bac); animalcell expression plasmids (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV,pcDNAI/Neo); bacteriophages such as λ phage; insect viral vectors suchas baculovirus (e.g., BmNPV, AcNPV); animal viral vectors such asretrovirus, vaccinia virus, adenovirus and adeno-associated virus; andthe like.

The promoter may be any promoter, as long as it is appropriate for thehost used to express the gene.

For example, when the host is an animal cell, useful promoters includepromoters derived from cytomegalovirus (CMV) (e.g., CMV immediate-earlypromoter), promoters derived from human immunodeficiency virus (HIV)(e.g., HIV LTR), promoters derived from Rous sarcoma virus (RSV) (e.g.,RSV LTR), promoters derived from mouse mammary cancer virus (MMTV)(e.g., MMTV LTR), promoters derived from Moloney murine leukemia virus(MoMLV) (e.g., MoMLV LTR), promoters derived from herpes simplex virus(HSV) (e.g., HSV thymidine kinase (TK) promoter), promoters derived fromSV40 (e.g., SV40 early promoter), promoters derived from Epstein-Barrvirus (EBV), promoters derived from adeno-associated virus (AAV) (e.g.,AAV p5 promoter), promoters derived from adenovirus (AdV) (Ad2 or Ad5major late promoter) and the like.

When the host is a bacterium of the genus Escherichia, the trp promoter,the lac promoter, the recA promoter, the XP_(L) promoter, the lpppromoter, the T7 promoter and the like are preferred.

When the host is a bacterium of the genus Bacillus, the SPOT promoter,the SPO2 promoter, the penP promoter and the like are preferred.

When the host is yeast, the PHO5 promoter, the PGK promoter, the GAPpromoter, the ADH promoter and the like are preferred.

When the host is an insect cell, the polyhedrin promoter, the P10promoter and the like are preferred.

Useful expression vectors include, in addition to the above, thoseoptionally harboring an enhancer, a splicing signal, a polyA additionsignal, a selection marker, an SV40 replication origin and the like. Asexamples of the selection marker, the dihydrofolate reductase (dhfr)gene [methotrexate (MTX) resistance], the ampicillin resistance(Amp^(r)) gene, the neomycin resistance (Neo^(r)) gene (G418 resistance)and the like can be mentioned. In particular, when a dhfr-deficientChinese hamster (CHO-dhfr⁻) cell is used in combination with the dhfrgene as the selection marker, a target gene can also be selected using athymidine-free medium.

Where necessary, a base sequence encoding a signal sequence (signalcodon) suitable for the host can be added to the 5′-end side of the DNAencoding HB-EGF or a partial peptide thereof. When the host is abacterium of the genus Escherichia, useful sequences include PhoA signalsequence, OmpA⋅signal sequence and the like; when the host is abacterium of the genus Bacillus, useful sequences include α-amylasesignal sequence, subtilising⋅signal sequence and the like; when the hostis yeast, useful sequences include MFα⋅signal sequence, SUC2⋅signalsequence and the like; and when the host is an animal cell, usefulsequences include insulin⋅signal sequence, α-interferon⋅signal sequence,antibody molecule⋅signal sequence and the like.

Useful hosts include, for example, a bacterium of the genus Escherichia,a bacterium of the genus Bacillus, yeast, an insect cell, an insect, ananimal cell and the like.

Useful bacteria of the genus Escherichia include, for example,Escherichia coli K12, DH1, JM103, JA221, HB101, C600 and the like.

Useful bacteria of the genus Bacillus include, for example, Bacillussubtilis MI114, 207-21 and the like.

Useful yeasts include, for example, Saccharomyces cerevisiae AH22,AH22R⁻, NA87-11A, DKD-5D and 20B-12, Schizosaccharomyces pombe NCYC1913and NCYC2036, Pichia pastoris KM71, and the like.

Useful insect cells include, for example, Spodoptera frugiperda cell (Sfcell), MG1 cell derived from the mid-intestine of Trichoplusia ni, HighFive™ cell derived from an egg of Trichoplusia ni, cell derived fromMamestra brassicae, cell derived from Estigmena acrea, and the like canbe mentioned when the virus is AcNPV. When the virus is BmNPV, usefulinsect cells include Bombyx mori N cell (BmN cell) and the like. UsefulSf cells include, for example, Sf9 cell (ATCC CRL1711), Sf21 cell (bothin Vaughn, J. L. et al., In Vivo, 13, 213-217 (1977) and the like.

Useful insects include, for example, a larva of Bombyx mori and thelike.

Useful animal cells include, for example, cell derived from a monkey(e.g., COS-1, COS-7, CV-1, Vero), cell derived from a hamster (e.g.,BHK, CHO, CHO-K1, CHO-dhfr⁻), cell derived from a mouse (e.g., NIH3T3,L, L929, CTLL-2, AtT-20), cell derived from a rat (e.g., H4IIE, PC-12,3Y1, NBT-II), cell derived from a human (e.g., HEK293, A549, HeLa,HepG2, HL-60, Jurkat, U937) and the like.

Transformation can be carried out according to the kind of host inaccordance with a publicly known method.

A bacterium of the genus Escherichia can be transformed, for example, inaccordance with a method described in Proc. Natl. Acad. Sci. U.S.A.,Vol. 69, 2110 (1972), Gene, Vol. 17, 107 (1982) and the like.

A bacterium of the genus Bacillus can be transformed, for example,according to a method described in Molecular and General Genetics, Vol.168, 111 (1979) and the like.

Yeast can be transformed, for example, in accordance with a methoddescribed in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc.Natl. Acad. Sci. USA, Vol. 75, 1929 (1978) and the like.

An insect cell and an insect can be transformed, for example, accordingto a method described in Bio/Technology, 6, 47-55 (1988) and the like.

An animal cell can be transformed, for example, in accordance with amethod described in Saibo Kogaku (Cell Engineering), extra issue 8, ShinSaibo Kogaku Jikken Protocol (New Cell Engineering ExperimentalProtocol), 263-267 (1995), published by Shujunsha, or Virology, Vol. 52,456 (1973).

HB-EGF can be separated and purified from the culture obtained bycultivating the aforementioned transformant according to a method knownper se.

For example, when HB-EGF is extracted from a cultured bacterium or acell cytoplasm, a method is used as appropriate wherein bacteria orcells are collected from a culture by a known means, suspended in anappropriate buffer solution, and disrupted by means of sonication,lysozyme and/or freeze-thawing and the like, after which a crude extractof soluble protein is obtained by centrifugation or filtration. Thebuffer solution may contain a protein denaturant such as urea orguanidine hydrochloride and a surfactant such as Triton X-100™ (when thesurfactant is contained, some or all of membrane proteins and organelleproteins can be simultaneously extracted). On the other hand, whenHB-EGF are extracted from a membrane fraction, a method is used whereinbacteria or cells are disrupted in the same manner as mentioned above,after which a debris of the cells is precipitated and removed bylow-speed centrifugation, a supernatant is subjected to high-speedcentrifugation to precipitate membrane-containing fraction (asnecessary, cellular membrane fraction, mitochondrial fraction, nucleusfraction and the like can be separated and purified by density gradientcentrifugation and the like), and the like. When HB-EGF are secreted outof bacteria (cells), a method is used wherein a culture supernatant issorted from a culture by centrifugation, filtration or the like, and thelike.

Isolation and purification of HB-EGF contained in the thus-obtainedsoluble fraction, membrane fraction or culture supernatant can beconducted according to a method know per se. Useful methods includemethods based on solubility, such as salting-out and solventprecipitation; methods based mainly on molecular weight differences,such as dialysis, ultrafiltration, gel filtration, andSDS-polyacrylamide gel electrophoresis; methods based on chargedifferences, such as ion exchange chromatography; methods based onspecific affinity, such as affinity chromatography; methods based onhydrophobicity differences, such as reversed-phase high performanceliquid chromatography; and methods based on isoelectric pointdifferences, such as isoelectric focusing electrophoresis. These methodscan be combined as appropriate.

When the thus-obtained HB-EGF or a partial peptide thereof is a freeform, it can be converted to a salt by a method known per se or a methodbased thereon; when the protein or peptide is obtained as a salt, it canbe converted to a free form or another salt by a method known per se ora method based thereon.

Note that HB-EGF produced by the transformant can also be optionallymodified by the action of an appropriate protein-modifying enzyme,before or after purification, or can have a polypeptide thereof removedpartially. As such, useful protein-modifying enzymes include, forexample, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase,glycosidase and the like.

The presence of the thus-obtained HB-EGF can be confirmed by enzymeimmunoassay, Western blotting and the like using an antibody specificthereto.

Furthermore, HB-EGF or a partial peptide thereof can also be synthesizedby in vitro translation using a cell-free protein translation systemcomprising a rabbit reticulocyte lysate, wheat germ lysate, Escherichiacoli lysate and the like, with RNA corresponding to the above-describedDNA encoding HB-EGF or a partial peptide thereof as the template.Alternatively, HB-EGF or a partial peptide thereof can be synthesizedusing a cell-free transcription/translation system further containingRNA polymerase, with the DNA encoding HB-EGF or a partial peptidethereof as the template. As the cell-free proteintranscription/translation system, commercially available one may beused, or may also be prepared by a method known per se; specifically,Escherichia coli extract can be prepared according to the methodsdescribed in Pratt, J. M. et al., Transcription and Translation, Hames,B. D. and Higgins, S. J. eds., IRL Press, Oxford 179-209 (1984), and thelike. As commercially available cell lysates, those derived fromEscherichia coli include E. coli S30 extract system (manufactured byPromega), RTS 500 Rapid Translation System (manufactured by Roche) andthe like; those derived from rabbit reticulocyte include RabbitReticulocyte Lysate System (manufactured by Promega) and the like: andfurthermore, those derived from wheat germ include PROTEIOS™(manufactured by TOYOBO) and the like. Of these, cell lysates using awheat germ lysate are preferable. Examples of the production method of awheat germ lysate include the methods described in Johnston, F. B. etal., Nature, 179: 160-161 (1957), Erickson, A. H. et al., Meth.Enzymol., 96: 38-50 (1996) and the like.

Useful systems or apparatuses for protein synthesis include the batchmethod (Pratt, J. M. et al. (1984) mentioned above), continuouscell-free protein synthesis system (Spirin, A. S. et al., Science, 242:1162-1164 (1988)) wherein amino acids, energy source and the like arecontinuously supplied to a reaction system, dialysis (Kigawa et al.,21st Annual Meeting of the Molecular Biology Society of Japan, WID6),the overlay method (instruction manual of PROTEIOS™ Wheat germ cell-freeprotein synthesis core kit: manufactured by TOYOBO) and the like. Usefulmethods additionally include one wherein template RNAs, amino acids,energy sources and the like are supplied to a synthesis reaction systemwhen needed, and synthetic substances and decomposed substances aredischarged when needed (JP-A-2000-333673), and the like.

HB-EGF exhibits liver protection (inhibition of liver damage) action,apoptosis (hepatocyte death) inhibitory action, liver regeneration(differentiation and/or division of hepatocyte)-inducing action and thelike; therefore, HB-EGF, or nucleic acids encoding HB-EGF or a partialpeptide thereof can be applied as an agent for protecting the liver, aninhibitor of hepatocyte apoptosis and a liver regeneration promoter, andcan be used for the prophylaxis/treatment of liver diseases such asliver damage and various diseases associated with hepatocyte death, forexample, acute liver damage (fulminant hepatic failure, acute hepatitis,drug-induced hepatitis), chronic hepatitis, autoimmune liver disease(autoimmune hepatitis, primary biliary cirrhosis), viral hepatitis (typeA-E), liver fibrosis, cirrhosis, liver carcinoma, alcoholic liverdisease, drug-induced liver disease (toxic drug-induced liver disease,allergic drug-induced liver disease), liver abscess, hepatic parasitosis(schistosomiasis Japonica, clonorchiasis), hepatic amyloidosis, lupoidhepatitis) and the like, in a hepatectomy, or in promoting liverregeneration after a liver transplantation.

(1) An Agent Containing HB-EGF for Protecting the Liver/Promoting LiverRegeneration

Since an agent for protecting the liver or promoting liver regenerationcontaining HB-EGF provides effect in several hours after administration,it can be preferably used for acute liver diseases, particularly acuteliver diseases accompanied by hepatocyte apoptosis due to inflammation.However, it can also be preferably used as an agent for theprophylaxis/inhibition of progression, which aims at preventingexhaustion of hepatocytes and cell death associated with chronicdiseases such as chronic hepatitis, liver fibrosis and cirrhosis by, asdescribed below, improving the stability by taking the dosage form of asustained release preparation or forming an immunoconjugate with anantibody.

HB-EGF can be used as it is, or may be mixed with a pharmacologicallyacceptable carrier as necessary to form a pharmaceutical composition andused as a pharmaceutical agent mentioned above.

Here, as examples of the pharmacologically acceptable carrier, variousorganic or inorganic carrier substances conventionally used aspharmaceutical preparation materials can be mentioned, and these areformulated as excipients, lubricants, binders and disintegrants, insolid preparations; as solvents, solubilizing agents, suspending agents,isotonizing agents, buffering agents and soothing agents, in liquidpreparations, and the like. Also, as necessary, pharmaceuticalpreparation additives such as antiseptics, antioxidants, colorants,sweeteners and the like can be used.

As examples of suitable excipients, lactose, saccharose, D-mannitol,D-sorbitol, starch, gelatinized starch, dextrin, crystalline cellulose,low substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose,gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminumsilicate, magnesium aluminometasilicate and the like can be mentioned.

As examples of suitable lubricants, magnesium stearate, calciumstearate, talc, colloidal silica and the like can be mentioned.

As examples of suitable binders, gelatinized starch, sucrose, gelatin,gum arabic, methyl cellulose, carboxymethyl cellulose, sodiumcarboxymethyl cellulose, crystalline cellulose, saccharose, D-mannitol,trehalose, dextrin, pullulan, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyvinyl pyrrolidone and the like can bementioned.

As examples of suitable disintegrants, lactose, saccharose, starch,carboxymethyl cellulose, calcium carboxymethyl cellulose, sodiumcrosscarmellose, sodium carboxymethyl starch, light anhydrous silicicacid, low substituted hydroxypropyl cellulose and the like can bementioned.

As examples of suitable solvents, water for injection, physiologicalsaline, Ringer's solutions, alcohols, propylene glycol, polyethyleneglycol, sesame oil, corn oil, olive oil, cottonseed oil and the like canbe mentioned.

As examples of suitable solubilizing agents, polyethylene glycol,propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate, sodium salicylate, sodium acetate and the like can bementioned.

As examples of suitable suspending agents, surfactants such as stearyltriethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid,lecithin, benzalkonium chloride, benzethonium chloride and glycerylmonostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose,hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropylcellulose; polysorbates, polyoxyethylene hardened castor oil and thelike can be mentioned.

As examples of suitable isotonizing agents, sodium chloride, glycerin,D-mannitol, D-sorbitol, glucose and the like can be mentioned.

As examples of suitable buffers, buffer solutions of a phosphate, anacetate, a carbonate, a citrate and the like, and the like can bementioned.

As examples of suitable soothing agents, benzyl alcohol and the like canbe mentioned.

As examples of suitable antiseptics, paraoxybenzoates, chlorobutanol,benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid andthe like can be mentioned.

As examples of suitable antioxidants, sulfides, ascorbates and the likecan be mentioned.

As examples of suitable colorant s, aqueous food tar colors (e.g., foodcolors such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and FoodBlue Nos. 1 and 2), water-insoluble lake pigments (e.g., aluminum saltsof the aforementioned aqueous food tar colors and the like), naturalpigments (e.g., β-carotene, chlorophyll, red iron oxide and the like)and the like can be mentioned.

As examples of suitable sweeteners, sodium saccharide, dipotassiumglycyrrhizinate, aspartame, stevia and the like can be mentioned.

As examples of dosage forms of the aforementioned pharmaceuticalcomposition, oral formulations such as tablets, capsules (including softcapsules and microcapsules), granules, powders, syrups, emulsions andsuspensions; non-oral formulations such as injections (e.g.,subcutaneous injections, intravenous injections, intramuscularinjections, intraperitoneal injections and the like), externalformulations (e.g., nasal preparations, transdermal preparations,ointments and the like), suppositories (e.g., rectal suppositories,vaginal suppositories and the like), pellets, drops, sustained-releasepreparations (e.g., sustained-release microcapsules and the like) andthe like can be mentioned.

The pharmaceutical composition can be produced by a methodconventionally used in the field of pharmaceutical preparation making,for example, a method described in the Japanese Pharmacopoeia and thelike. A specific method of producing a preparation is hereinafterdescribed in detail. The content of active ingredient in thepharmaceutical composition varies depending on the dosage form, the doseof the active ingredient and the like; and is, for example, from about0.1 to 100% by weight.

For example, an oral formulation is produced by adding to an activeingredient an excipient (e.g., lactose, saccharose, starch, D-mannitoland the like), a disintegrant (e.g., calcium carboxymethyl cellulose andthe like), a binder (e.g., gelatinized starch, gum arabic, carboxymethylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone and the like),a lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000and the like) and the like, compression molding the resultant mixture,and subsequently, as required, coating the resulting material with acoating base by a method known per se for the purpose of taste masking,enteric solubility or sustained release.

As examples of the coating base, a sugar-coating base, a aqueous filmcoating base, an enteric film coating base, a sustained-release filmcoating base and the like can be mentioned.

As the sugar-coating base, saccharose is used, which may be used incombination with one species or two or more species selected from amongtalc, precipitated calcium carbonate, gelatin, gum arabic, pullulan,carnauba wax and the like.

As examples of the aqueous film coating base, cellulose polymers such ashydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethylcellulose and methylhydroxyethyl cellulose; synthetic polymers such aspolyvinylacetal diethylanimoacetate, aminoalkylmethacrylate copolymer E[Eudragit-E (trade name), Rohm Pharma Corp.] and polyvinyl pyrrolidone;polysaccharides such as pullulan; and the like can be mentioned.

As examples of the enteric film coating base, cellulose polymers such ashydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl celluloseacetate succinate, carboxymethylethyl cellulose, and cellulose acetatephthalate; acrylic polymers such as Methacrylic Acid Copolymer L[Eudragit-L (trade name), Rohm Pharma Corp.], Methacrylic Acid CopolymerLD [Eudragit-L-30D55 (trade name), Rohm Pharma Corp.], and MethacrylicAcid Copolymer S [Eudragit-S (trade name), Rohm Pharma Corp.]; naturalsubstances such as shellac, and the like can be mentioned.

As examples of the sustained-release film coating base, cellulosepolymers such as ethyl cellulose; acrylic polymers such as aminoalkylmethacrylate copolymer RS [Eudragit-RS (trade name), Rohm Pharma Corp.],and an ethyl acrylate-methylmethacrylate copolymer suspension[Eudragit-NE (trade name), Rohm Pharma Corp.]; and the like can bementioned.

The above-mentioned coating bases may also be used in a mixture of twoor more kinds thereof in a suitable ratio. Also, during coating, ashading agent, for example, titanium oxide or iron sesquioxide, may beused.

Examples of preparations suitable for parenteral administration (e.g.,subcutaneous injection, intramuscular injection, topical injection,intraperitoneal administration and the like) include aqueous andnonaqueous isotonic aseptic injection liquids, in which antioxidant,buffer, antiviral agent, isotonicity agent and the like can becontained. The examples also include aqueous and nonaqueous asepticsuspension liquids optionally containing suspension agent, solubilizer,thickener, stabilizer, preservative and the like. When theadministration method is a topical injection around a target region,injectable liquids are preferable. Alternatively, a sustained-releasepreparation can be prepared by using a bioaffinity material such ascollagen. Since the Pluronic gel turns into a gel at the bodytemperature, and is present in a liquid state at a temperature nothigher than the body temperature, HB-EGF can be topically injected alongwith a Pluronic gel and gelatinized around a target tissue to achievelong-term sustainability. The protein preparation can be sealed in acontainer by a unit dose or plural doses like ampoules and vials. Inaddition, HB-EGF and a pharmaceutically acceptable carrier can belyophilized and preserved such that they only need to be dissolved orsuspended in a suitable aseptic vehicle immediately before use.

Since antibodies against a hepatocyte surface molecule can specificallydeliver a pharmaceutical agent into a hepatocyte, stability of HB-EGF inblood and efficiency in delivery can be improved by preparing animmunoconjugate in which HB-EGF are crosslinked with the antibody.Examples of the hepatocyte surface molecules include, but not limitedto, EGFR (HER1), HER2, HER3, HER4 and the like. When an anti-EGFRantibody is used, a nonneutralizing antibody is preferably used as anantibody for targeting so as not to inhibit signal transduction fromEGFR.

Although the antibody against a hepatocyte surface molecule may be apolyclonal antibody or monoclonal antibody, a monoclonal antibody ispreferred. The antibody can be produced by a well known immunologicaltechnique. The antibody may be a complete antibody molecule or afragment. The fragment may be any, as long as it possesses an antigenbinding site (CDR) to the hepatocyte surface molecule; examples includeFab, F(ab′)₂, ScFv, minibody and the like.

The monoclonal antibody can be produced by cell fusion method (e.g.,Takeshi Watanabe, Saiboyugoho no Genri to Monoclonal-Kotai no Sakusei,Akira Taniuchi, edited by Toshitada Takahashi, “Monoclonal-Kotai toGan-Kiso to Rinsho-”, 2-14, Science Forum Publishing, (1985)). Forexample, a mouse is administrated subcutaneously or intraperitoneally 2to 4 times with a target protein (as necessary, the protein can becrosslinked with a carrier protein such as bovine serum albumin and KLH(Keyhole Limpet Hemocyanin) to form a complex) being an antigen alongwith a commercially available adjuvant, a spleen or lymph node iscollected about 3 days after the final administration to collectleukocytes. The leukocytes and myeloma cells (e.g., NS-1, P3X63Ag8 andthe like) are subjected to a cell fusion to give hybridomas that producea monoclonal antibody against the factor. The cell fusion may beperformed by the PEG method [J. Immunol. Methods, 81(2): 223-228 (1985)]or by the voltage pulse method [Hybridoma, 7(6): 627-633 (1988)]. Ahybridoma that produces the desired monoclonal antibody can be selectedby detecting an antibody that binds specifically to the antigen from theculture supernatant using a widely known EIA or RIA method and the like.Cultivation of the hybridoma that produces the monoclonal antibody canbe performed in vitro, or in vivo such as in mouse or rat, preferably inmouse ascitic fluid, and the antibody can be acquired from the culturesupernatant of the hybridoma and the ascitic fluid of the animal,respectively.

Considering administration to a human, however, the antibody ispreferably a chimeric antibody of a human and another animal (e.g.,mouse and the like), more preferably a humanized antibody, and mostpreferably a complete human antibody. “Chimeric antibody” herein refersto an antibody having an immunized animal-derived variable region (Vregion) and a human-derived constant region (C region), and “humanizedantibody” refers to an antibody wherein all regions other than CDR arereplaced with a human antibody. A chimeric antibody and humanizedantibody can be acquired by, for example, cleaving out the sequenceencoding the V region or CDR from a mouse monoclonal antibody geneprepared by the same method as described above, cloning a chimeric geneprepared by fusion of the sequence with the DNA encoding the C region ofa human myeloma-derived antibody into an appropriate expression vector,and introducing this into an appropriate host cell to allow the chimericgene to be expressed. A complete human antibody can be acquired by usinga known phage display library, or by using a human antibody-producingmouse developed by Medarex, Inc. or a KM mouse jointly-developed byMedarex, Inc. and Kirin Inc.

Useful methods of crosslinking of an antibody against a hepatocytesurface molecule and HB-EGF include, but not limited to, the methoddescribed in Adv. Drug Deliv. Rev., 53: 171-216 (2001).

An agent containing HB-EGF for protecting the liver/promoting liverregeneration, which has been designed to have the dosage form mentionedabove, can be dissolved or suspended in a suitable aseptic vehicle andadministered orally or parenterally to, for example, mammals (e.g.,human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and thelike). Examples of the parenteral administration route includeintravenous, intraarterial, intramuscular, intraperitoneal andintratracheal routes and the like.

The dose of the agent containing HB-EGF for protecting theliver/promoting liver regeneration varies depending on the molecularweight of the active ingredient, administration route, severity ofdisease, animal species to be the subject of administration, drugacceptability, body weight, age and the like of the subject ofadministration; generally, the dose is within the range of about 0.05 toabout 10 mg/kg, preferably about 0.1 to about 5 mg/kg, as the amount ofthe active ingredient, per day for an adult, which may be administeredat once or in several portions.

(2) Agent Containing Nucleic Acid Encoding HB-EGF for Protecting theLiver/Promoting Liver Regeneration

The nucleic acid encoding HB-EGF can produce HB-EGF for a long perioddue to prolonged gene expression and, therefore, is expected to exhibita sustained treatment effect. Therefore, the nucleic acid can also bepreferably used, in addition to the prophylactic or therapeutic effecton acute liver diseases, as an agent for the prophylaxis/inhibition ofprogression, which is used for preventing exhaustion of hepatocytes andcell death associated with chronic diseases such as chronic hepatitis,liver fibrosis and cirrhosis.

The nucleic acid encoding HB-EGF is preferably administered in the formwherein it is carried on a suitable expression vector. The expressionvector is configured at the position where the nucleic acid encodingHB-EGF is functionally linked to a promoter capable of exhibitingpromoter activity in a target cell of a mammal, which is the subject ofadministration, or can be turned into a functionally-linked form in atarget cell of the animal administered under given conditions. Thepromoter to be used is not particularly limited as long as it canfunction in a target cell of a mammal, which is the subject ofadministration. Examples of the promoter include virus promoters such aspromoters derived from cytomegalovirus (CMV) (e.g., CMV immediate-earlypromoter), promoters derived from human immunodeficiency virus (HIV)(e.g., HIV LTR), promoters derived from Rous sarcoma virus (RSV) (e.g.,RSV LTR), promoters derived from mouse mammary tumor virus (MMTV) (e.g.,MMTV LTR), promoters derived from Moloney mouse leukemia virus (MoMLV)(e.g., MoMLV LTR), promoters derived from herpes simplex virus (HSV)(e.g., HSV thymidine kinase (TK) promoter), promoters derived from SV40(e.g., SV40 early promoter), promoters derived from Epstein-Barr virus(EBV), promoters derived from adeno-associated virus (AAV) (e.g., AAV p5promoter), and promoters derived from adenovirus (AdV) (Ad2 or Ad5 majorlate promoter), and constitutive protein gene promoters in mammal suchas (3-actin gene promoter, PGK gene promoter, transferrin gene promoter,and the like. “Being configured at the position where the expressionvector can be turned into a functionally-linked form under a constantcondition” means, for example, that as further described below, theexpression vector has a structure where the promoter and the nucleicacid encoding HB-EGF are divided by two recombinase recognitionsequences configured at the same direction, wherein the two recombinaserecognition sequences are separated by a spacer sequence that has asufficient length to prevent the expression of the nucleic acid from thepromoter, the spacer sequence is cleaved out in the presence of arecombinase that specifically recognizes the recognition sequence, andthe nucleic acid encoding HB-EGF is configured so as to be functionallylinked to the promoter.

The expression vector preferably contains a transcription terminationsignal, i.e. terminator region, in the downstream of the nucleic acidencoding HB-EGF. The expression vector can further contain a selectionmarker gene for selection of transformed cells (genes that offerresistance against pharmaceutical agents such as tetracycline,ampicillin, kanamycin, hygromycin, and phosphinothricin, genes thatcomplement an auxotrophic mutation, and the like). When the expressionvector has a spacer sequence sandwiched by recombinase recognitionsequences as mentioned above, the selection marker gene can beconfigured within the spacer sequence.

Although vector used as the expression vector of the present inventionis not particularly limited, examples of vectors suitable foradministration into a mammal such as human include vectors derived fromvirus such as vretrovirus, adenovirus, adeno-associated virus,herpesvirus, herpes simplex virus, lentivirus, vaccinia virus, poxvirus,poliovirus, sindbisvirus, and Hemagglutinating Virus of Japan.Adenovirus has advantages that it has extremely high efficiency of geneintroduction, permits introduction into a nondividing cell, theintegration of the introduced gene into a host chromosome is extremelyrare, and the like. Particularly, development of next-generation vectornamed gutted (gutless) vector, wherein almost full-length of adenovirusgenome other than packaging signal (ψ) is substituted by introducedgene, has resolved the problem of immunogenicity in first-generationvectors, and thereby long-term sustainability of introduced geneexpression has been realized, which results in further increase ofusability of adenovirus in gene therapy. Similarly, sinceadeno-associated virus has comparatively high efficiency of geneintroduction, permits introduction into a nondividing cell includinghepatocyte, and it has been known from animal experiments thatexpression of introduced gene on administration into a living organismpersists over a long period, adeno-associated virus is preferred as theviral vector in the present invention.

It is possible that constitutive overexpression of HB-EGF causes a sideeffect in an animal into which the gene has been introduced.Accordingly, in a preferable embodiment of the present invention, theexpression vector is capable of expressing HB-EGF in a time- and/ortarget cell-specific manner in order to prevent adverse influences dueto an overexpression of HB-EGF during the period and/or at a site not inneed thereof. As the first embodiment of such vector, vectors containinga nucleic acid encoding HB-EGF functionally linked to a promoter derivedfrom a gene that is specifically expressed in a target cell (in thepresent invention, the target cell is preferably a hepatocyte, but isnot limited as long as the target cell can release soluble HB-EGF todeliver same to the liver) of an animal to be the subject ofadministration can be mentioned. For example, as the liver specificpromoters, serum albumin promoter, cytochrome P-450 promoter, andpromoters containing a liver-specific transcription factor (HNF1, HNF3,HNF4, C/EBP, and the like)-binding cis-element, and the like can bementioned.

As the second embodiment of the time- and tissue-specific expressionvector of the present invention, vectors containing a nucleic acidencoding HB-EGF functionally linked to an inducible promoter whoseexpression is trans-regulated by an exogenous substance can bementioned. When the inducible promoter used is, for example,metallothionein-1 gene promoter, expression of HB-EGF can be induced atany time in a target cell-specific manner by administering topically toa location of target cell an inducing substance, including heavy-metalssuch as gold, zinc and cadmium, steroids such as dexamethasone,alkylating agents, chelating agents, cytokines and the like, at intendedtime.

Another preferable embodiment of the time- and tissue-specificexpression vector of the present invention is a vector having astructure wherein a promoter and a nucleic acid encoding HB-EGF aredivided by a spacer sequence having a sufficient length to preventexpression of nucleic acid from a promoter, i.e., by two recombinaserecognition sequences configured in the same direction. Mereintroduction of the vector into a target cell is insufficient for apromoter to direct transcription of nucleic acid encoding HB-EGF.However, when a recombinase specifically recognizing the recognitionsequence at a desired timing is topically administered to a target cell,or an expression vector containing a nucleic acid encoding therecombinase is topically administered to allow expression of therecombinase in the target cell, homologous recombination via therecombinase takes place between the recognition sequences, as a resultof which the spacer sequence is cleaved out, the nucleic acid encodingHB-EGF is functionally linked to the promoter and a target cell specificexpression of HB-EGF occurs at a desired timing.

To prevent recombination by a recombinase endogenous in the subject ofadministration, the recombinase recognition sequence to be used for theabove-mentioned vector is desirably a heterologous recombinaserecognition sequence which is not recognized by the endogenousrecombinase. Accordingly, the recombinase acting trans on the vector isalso desirably a heterologous recombinase. The combination of suchheterologous recombinase and the recombinase recognition sequencepreferably includes, nonlimitatively, Escherichia coli bacteriophageP1-derived Crerecombinase and lox Psequence, or yeast-derivedFlprecombinase and frtsequence.

As a promoter for the time specific and tissue specific expressionvector of the present invention utilizing the interaction betweenrecombinase/recombinase recognition sequences, a virus-derived promoteror a promoter of a constituent protein gene of a mammal is preferablyused to ensure expression at a desired timing and site.

The expression vector containing a nucleic acid encoding HB-EGF can beproduced by using a conventional genetic engineering technique, cellculturing technique and virus preparation technique [for example,Current Protocols in Molecular Biology, F. Ausubel et al. eds. (1994)John Wiley & Sons, Inc.; Molecular Cloning (A Laboratory Manual), 3rded. Volumes 1-3, Josseph Sambrook & David W. Russel eds., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y.) (2001); Culture ofAnimal Cells; A Manual of Basic Technique, R. Freshney eds., 2nd ed.(1987), Wiley-Liss; Frank L. Graham, Manipulation of adenovirus vector,Chapter 11. p 109-p 128; E. J. Murray eds., Methods in MolecularBiology, Vol. 7, Gene Transfer and Expression Protocols (1991); Chen,S-H. et al., Combination gene therapy for liver metastases of coloncarcinoma in vivo, Proc. Natl. Acad. Sci. USA (1995) 92, 2477-2581, andthe like].

When a nonviral vector is used as the expression vector containing anucleic acid encoding HB-EGF, introduction of the expression vector canbe performed by using a polymer carrier such as poly-L-lysine-nucleicacid complex, or by encapsulating the vector in a liposome. The liposomeis a capsule composed of phospholipid with a particle size of severaltens to several hundreds nm, inside of which vectors such as a plasmidencoding HB-EGF can be encapsulated. Alternatively, vectors can bedirectly introduced into a target cell by using the particle gun method.

In the use of the vector in which the interaction between a recombinaseand a recombinase recognition sequence is utilized, when the recombinaseper se is topically administered as a trans-acting substance, forexample, the recombinase can be dissolved or suspended in a suitableaseptic vehicle and injected into a target site. On the other hand, whena recombinase-expressing vector is topically administered to a targetsite as a trans-acting substance, the recombinase-expressing vector isnot particularly limited as long as the nucleic acid encoding therecombinase has a expression cassette that is functionally linked to apromoter capable of exhibiting a promoter activity in a target cell ofsubject of administration. When the promoter used is a constitutivepromoter, in order to prevent the expression of the recombinase duringthe period not in need thereof, the vector administered to a target siteis preferably one rarely incorporated into a chromosome of host cell,including, for example, adenovirus. As an alternative approach forallowing the expression of recombinase at an intended time, the use ofan inducible promoter such as metallothionein gene promoter can bementioned. In this case, viral vectors with high integration efficiencysuch as retrovirus can be used.

Also, the expression vector containing a nucleic acid encoding HB-EGFcan be, where necessary, mixed with a pharmacologically acceptablecarrier and formulated into various forms of preparation such asinjection and the like, and used as the above-mentioned pharmaceuticalagent. Here, examples of the pharmacologically acceptable carrierinclude, but are not limited to, those mentioned above as preparationscontaining HB-EGF.

An agent containing an expression vector containing a nucleic acidencoding HB-EGF for protecting the liver/promoting liver regeneration isadministered by either ex vivo method wherein the target cell of thetreatment target animal itself or a cell from an animal (allogeneic orheterologous to the treatment target animal) is extracted out of thebody, cultured, subjected to infection, and put back (or transplanted)into the body, or in vivo method wherein the vector is directlyadministered into the body of the subject of administration to performintroduction. In the case of the ex vivo method, the introduction of thevector into the target cell can be performed by the microinjectionmethod, calcium phosphate coprecipitation method, PEG method,electroporation method and the like. In the case of the in vivo method,the administration of the preparation can be performed by, for example,injection, catheter, balloon catheter and the like.

Dose of the agent for protecting the liver/promoting liver regenerationof the present invention containing the expression vector containing thenucleic acid encoding HB-EGF varies depending on the kind of vector,size of active ingredient molecule, promoter activity, administrationroute, severity of illness, the animal species to be the subject ofadministration, drug acceptability, body weight, age and the like of thesubject of administration. For example, when adenovirus is used as theviral vector, since the safety was confirmed using 2×10¹¹ particles/kg(virus particles) in a clinical test of conventional gene therapy forliver diseases, this amount can be used as a rough standard dose. Forexample, the dose is about 2×10⁹ to about 2×10¹¹ particles/kg,preferably about 2×10¹⁰ to about 2×10¹¹ particles/kg, for an adult perday. Note that when HB-EGF is actually administered, HB-EGF gene doesnot need to be introduced into all hepatocytes since the liver diseasesare not congenital diseases and an amount not more than this level isconsidered to be sufficient for practical purposes. On the other hand,when a nonviral vector is encapsulated in a liposome, since the safetywas confirmed by intravenous administration of 666 μg of DNA in aclinical research using a cynomolgus with a body weight of about 4 kg,this amount can be used as a rough standard dose. For example, thesingle dose for an adult is about 2 to about 10 mg, preferably about 5to about 8 mg.

The present invention also intends liver protection and promotion ofliver regeneration, and further, prophylaxis/treatment of liverdiseases, by potentiating expression of endogenous HB-EGF gene andactivity of HB-EGF. As the means for potentiating the expression ofendogenous HB-EGF gene, for example, administration of a transactivationfactor, which binds to a regulate region of HB-EGF gene to activate thetranscription thereof, a substance that stabilizes HB-EGF mRNA, and asubstance that increases translation efficiency from HB-EGF mRNA, andthe like can be mentioned; as the means for potentiating the activity ofendogenous HB-EGF, for example, administration of a substance thatpromotes production of active form of soluble HB-EGF from pro-HB-EGF(e.g., specific proteases that are involved in shedding such as ADAM,and the like), a substance that suppresses the decomposition of HB-EGF(e.g., protease inhibitors and the like), and the like can be mentioned.

Accordingly, another aspect of the present invention provides ascreening method of substances for protecting the liver/promoting liverregeneration, comprising selecting the above-mentioned substance thatenhances the expression of endogenous HB-EGF gene and the activity ofHB-EGF. The screening method is characterized by culturing a cell thatexpresses HB-EGF by nature in the presence of and in the absence of atest substance, and comparing expression amount and/or activity ofHB-EGF. The expression amount of HB-EGF can be examined attranscriptional level by using Northern blot or RT-PCR, or attranslational level by immunoassay using an anti-HB-EGF antibody and thelike. On the other hand, the activity of HB-EGF can be examined byinvestigating the growth-stimulating activity in a cell such ashepatocyte, or by using activation of EGFR family (phosphorylation ofthe receptor, and the like), which is a target receptor of HB-EGF, oractivation of a kinase molecule such as MAPK, which is activated at thedownstream of EGFR family, as an index.

The thus selected substance mentioned above, which is capable ofenhancing expression of endogenous HB-EGF gene and activity of HB-EGF,can be prepared, for example, as a pharmaceutical composition along witha pharmacologically acceptable carrier in the same manner as mentionedabove for the agent containing HB-EGF for protecting the liver/promotingliver regeneration, and administered orally or parenterally to a mammalsuch as human as an agent for protecting the liver/promoting liverregeneration.

Dose of the agent containing the substance for protecting theliver/promoting liver regeneration varies depending on kind andmolecular weight of active ingredient, administration route, severity ofillness, the animal species to be the subject of administration, drugacceptability, body weight, age and the like of the subject ofadministration; generally, the dose is within the range of about 0.001to about 100 mg/kg, preferably about 1 to about 10 mg/kg, as the amountof an active ingredient, per day for an adult, which can be administeredat once or in several portions.

Abbreviations for bases, amino acids and the like used herein are basedon abbreviations specified by the IUPAC-IUB Commission on BiochemicalNomenclature or abbreviations in common use in relevant fields. Someexamples are given below. When an enantiomer may be present in aminoacid, it is of the L-configuration, unless otherwise stated.

-   DNA: Deoxyribonucleic acid-   cDNA: Complementary deoxyribonucleic acid-   A: Adenine-   T: Thymine-   G: Guanine-   C: Cytosine-   RNA: Ribonucleic acid-   mRNA: Messenger ribonucleic acid-   dATP: Deoxyadenosine triphosphate-   dTTP: Deoxythymidine triphosphate-   dGTP: Deoxyguanosine triphosphate-   dCTP: Deoxycytidine triphosphate-   ATP: Adenosine triphosphate-   EDTA: Ethylenediaminetetraacetic acid-   SDS: Sodium dodecyl sulfate-   Gly: Glycine-   Ala: Alanine-   Val: Valine-   Leu: Leucine-   Ile: Isoleucine-   Ser: Serine-   Thr: Threonine-   Cys: Cysteine-   Met: Methionine-   Glu: Glutamic acid-   Asp: Aspartic acid-   Lys: Lysine-   Arg: Arginine-   His: Histidine-   Phe: Phenylalanine-   Tyr: Tyrosine-   Trp: Tryptophan-   Pro: Proline-   Asn: Asparagine-   Gln: Glutamine-   pGlu: Pyroglutamic acid-   Me: Methyl group-   Et: Ethyl group-   Bu: Butyl group-   Ph: Phenyl group-   TC: Thiazolidine-4(R)-carboxamide group

Substituents, protecting groups and reagents frequently mentioned hereinare represented by the symbols shown below.

-   Tos: p-Toluenesulfonyl-   CHO: Formyl-   Bzl: Benzyl-   Cl₂Bzl: 2,6-Dichlorobenzyl-   Bom: Benzyloxymethyl-   Z: Benzyloxycarbonyl-   Cl—Z: 2-Chlorobenzyloxycarbonyl-   Br—Z: 2-Bromobenzyloxycarbonyl-   Boc: t-Butoxycarbonyl-   DNP: Dinitrophenol-   Trt: Trityl-   Bum: t-Butoxymethyl-   Fmoc: N-9-Fluorenylmethoxycarbonyl-   HOBt: 1-Hydroxybenztriazole-   HOOBt: 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-   HONB: 1-Hydroxy-5-norbornene-2,3-dicarboximide-   DCC: N,N′-Dicyclohexylcarbodiimide

The present invention is explained in more detail in the following byreferring to examples, which are mere examples and do not limit thescope of the present invention in any way. In the examples, geneticengineering techniques and cell culturing techniques handling plasmids,DNA, various enzymes, Escherichia coli, cultured cells and the like wereperformed according to the methods described in the above-mentionedCurrent Protocols in Molecular Biology, F. Ausubel et al. eds. (1994)John Wiley & Sons, Inc.; Molecular Cloning (A Laboratory Manual), 3rded. Volume 1-3, Josseph Sambrook & David W. Russel eds., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y.) (2001); Culture ofAnimal Cells; A Manual of Basic Technique, R. Freshney eds., 2nd ed.(1987) Wiley-Liss, unless otherwise stated. Unless otherwise stated,general handling of adenovirus was performed according to the methodsdescribed in the above-mentioned Frank L. Graham, Manipulation ofadenovirus vector, Chapter 11, p 109-p 128; E. J. Murray eds., Methodsin Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols(1991); Chen, S-H. et al., Combination gene therapy for liver metastasesof colon carcinoma in vivo, Proc. Natl. Acad. Sci. USA (1995) 92,2477-2581. Regarding therapeutic effects and phenomena, a significantdifference among any groups was first analyzed by Anova test, andsubsequently an individual significant difference between each two groupwas analyzed by Student t-test (asymmetric two-group t-test). Asignificant difference in survival rate was analyzed using Kaplan-Meiertest.

EXAMPLES Example 1 Construction of Adenoviral Vector

Adenoviral vectors used in Experimental Examples described below wereproduced as the following.

Plasmid pADL.1/RSV (B. Fang et al., Gene Therapy (1994) 1, 247-254) wasa plasmid produced by incorporating, from upstream, a 0-455 base portionfrom 3′ side of human adenovirus type 5, Rous sarcoma virus (RSV) LTRpromoter, multicloning site, poly A signal sequence of bovine growthhormone, and a 3328-5788 base portion from 3′ side of human adenovirustype 5 into pBR322 plasmid, and offered from Shu-Hsia Chen (Mount SinaiUniversity). The pADL.1/RSV plasmid was digested with restrictionenzymes Hind III and Not I and purified to give a vector to be used forligation. On the other hand, plasmid pRcHBEGF, containing cDNA of thefull length of ORF of human HB-EGF in plasmid pRc/CMV (Invitrogen), wasoffered from Eisuke Mekada (Research Institute for Microbial Diseases,Osaka University). The pRcHBEGF plasmid was digested with restrictionenzymes Hind III and Not I to excise the full length of HB-EGF cDNA,which was subjected to agarose gel electrophoresis, and the intended DNAfragment was recovered from the gel and purified to give an insert to beused for ligation. The thus treated pADL.1/RSV vector and HB-EGF cDNAinsert were subjected to a ligation reaction using T4 DNA ligase toobtain pADL.1/RSV-HB-EGF. Furthermore, the pADL.1/RSV-HB-EGF was,together with plasmid pJM17 (Micorobix Biosystems Inc.) containing agene other than μl region of human adenovirus type 5, co-transfectedinto 293 cell by calcium phosphate method. This caused a plaquecontaining the correct intended adenovirus to emerge by homologousrecombination 10 to 14 days after the co-transfection. This plaque waspicked up, and the correct non-proliferating recombinant adenovirusAd.HB-EGF expressing the intended HB-EGF was confirmed by immunostainingusing an anti-HB-EGF antibody (M-18:sc-1414, SANTA CRUZ), and the like,after which the Ad.HB-EGF was proliferated by 293 cell, and concentratedby density gradient centrifugation in cesium chloride. The concentratedvirus was purified with an Econo-Pac 10DG desalting column (Bio-Rad Cat.No. 732-2011), eluted with PBS(−), added with glycerol, frozen in liquidnitrogen, and preserved at −80° C. When the virus was used, the particlevolume was calculated, the solution was diluted with PBS(−) so as toachieve an administered volume of 100 μl/animal, and the dilutedsolution was administered.

A recombinant adenovirus AD.LacZ expressing LacZ gene of Escherichiacoli used for confirmation of gene introduction was produced by the samemethod as described above; details of the Ad.LacZ production method aredescribed in Proc. Natl. Acad. Sci. USA (1995) 92, 2577-2581. HB-EGF isnot incorporated into Ad.dE1.3, which is, therefore, a recombinantadenovirus not expressing these genes at all. Ad.dE1.3 was produced byco-transfecting pADL1/RSV not inserted by HB-EGF and pJM17 into 293 cellas mentioned above and following the same method and the process. HumanHB-EGF cDNA is described in Higashiyama, S. et al., Science 251: 936-939(1991), and Gene Bank accession number thereof is M60278 (the samesequence as NM_001945).

Experimental Example 1 Confirmation of Gene Introduction into Liver byAdenoviral Vector

Adenoviral vector Ad.LacZ and Ad.HB-EGF (1×10¹¹ particles) prepared inExample 1 were injected into 6-week-old male C57BL/6J mice (CHUBUSCIENCE Co., Ltd.; 10 animals per group) from the tail vein, and theexpressions of exogenous LacZ and HB-EGF in the liver were examined byX-gal staining and immunostaining, respectively. The mice were givengeneral anesthesia with ether, and their chest was opened, and theirorgans (heart, lung, liver, kidney, spleen) were collected. After thecollection, the organs were equally divided into two; one was used forpreparing an OCT specimen with a compound (TissueTek OCT compound), andanother was, after fixation with 10% formaldehyde, used for preparing aparaffin-embedded specimen. X-gal staining was performed by fixing OCTspecimen section with 0.2% formaldehyde/0.02% glutaraldehyde fixativefor 30 min, followed by immersion in X-gal staining solution and areaction at 37° C. for 24 hr. To perform immunostaining, theparaffin-embedded specimen was fixed with 4% para-formaldehyde for 10min, blocked with 10% skim milk (Snow Brand Milk Products Co., Ltd.) for60 min; subsequently, reacted with a primary antibody (anti-human HB-EGFgoat polyclonal antibody, R&D Systems Inc., Minneapolis, Minn. Cat. No.AF-259-NA) 100-fold diluted solution (2 μg/ml) for 1 hr, andvisualization of HB-EGF was performed by labeling same with anti-goatAlexa568 (MOLECULAR PROBES, Inc., Eugene, Oreg. Cat. No. A-11029).Nuclear staining was performed with a 1000-fold diluted Hoechst33342(MOLECULAR PROBES, Inc., Eugene, Oreg. Cat. No. H-3570) for 5 min.Recording of observed images was performed with a confocal lasermicroscope (Carl Zeiss product number LSM510). Results of X-gal stainingand immunostaining are shown in FIG. 1 with a sample which was injectedwith Ad.dE1.3 from the tail vein and treated in the same manner being anunstained negative control.

As FIG. 1 shows, since X-gal staining was observed in 60 to 90% ofhepatocytes from livers of mice which had been injected with Ad.LacZfrom the tail vein, it was demonstrated that tail vein injection ofadenoviral vector enabled efficient gene introduction into a hepatocyte(FIG. 1A). Furthermore, in mouse livers into which Ad.HB-EGF wasinjected from the tail vein, mainly expression of HB-EGF in cellularmembrane was observed and, hence, it was demonstrated that an exogeneousHB-EGF with biological activity was synthesized as a membrane-bindingtype HB-EGF, or a further processed soluble HB-EGF acted on a hepatocytein an autocrine manner (FIG. 1B).

Experimental Example 2 Inhibitory Effect of Elevation in BloodConcentration of Liver Enzyme by Ad.HB-EGF

6-week-old male C57BL/6J mice (CHUBU SCIENCE Co., Ltd.; 10animals/group) were injected with a Fas agonist antibody (anti-mouse Fasmouse monoclonal antibody, clone name, Jo-2, Becktone-DickinsonBioscience, San Jose, Calif. Cat. No. 554255) from the tail vein at 4 μgper animal to prepare a Fas-induced fulminant hepatic failure model. Toinvestigate the effect of prevention and treatment of fulminant hepaticfailure, the adenoviral vectors (Ad.HB-EGF, Ad.HGF, Ad.LacZ, andAd.dE1.3) were previously injected from the tail vein at 1×10¹¹particles per animal 72 hr before the injection of the Fas agonistantibody from the tail vein. Blood samples were collected at 24 hr and36 hr after the tail vein injection of the Fas agonist antibody (96 hrand 108 hr after the tail vein injection of adenovirus, respectively),the mouse were sacrificed, and the organs were collected (theexperimental schedule is shown in FIG. 2A). Liver function, which is anindex of prevention of onset and treatment of fulminant hepatic failure,was evaluated by measuring alanine aminotransferase (ALT) and asparticacid aminotransferase (AST), which are liver enzymes in liver. Themeasurement was carried out using an automatic clinical analyzer Hitachi736 (Hitachi, Ltd.) according to a conventional method.

As shown in FIGS. 2B and 2C, the results of measuring the serum ALT andAST level after the blood sampling demonstrated that at 24 hr after thetail vein injection of the agonist Fas antibody, while the levels of ALTand AST were elevated to 2240±450 and 1665±391 IU/L, respectively, inthe Ad.dE1.3-administered group, both the levels were not more than 230IU/L in the Ad.HB-EGF-administered group, showing inhibition ofelevation in ALT and AST level. Furthermore, it was demonstrated that inthis inhibitory effect of elevation in liver enzyme in blood, HB-EGFshowed the equivalent effect to HGF, only which had been known to showstrong effect of prevention and treatment of fulminant hepatic failure.It was also demonstrated that at 36 hr after administration of antibody,ALT and AST level were decreased to not more than 500 IU/L even in theAd.dE1.3-administered group; while in the Ad.HGF-administered group,there observed no significant difference with this, in theAd.HB-EGF-administered group, the levels were not more than 250 IU/L,which were significantly lower than that in the Ad.dE1.3-administeredgroup. From the above results, it was demonstrated that Ad.HB-EGFinhibited and relieved liver damage associated with fulminant hepaticfailure, and the effect was more potent than that of Ad.HGF.

Experimental Example 3 Inhibitory Effect of Hepatocyte Apoptosis byAd.HB-EGF

Hepatocyte death due to apoptosis, which is a main pathology offulminant hepatic failure, was evaluated by performing HE staining andTUNEL staining of liver tissues. Sections were respectively preparedfrom the paraffin-embedded specimen and OCT specimen of mouse livers at24 hr and 36 hr after the agonist antibody administration (96 hr and 108hr after the tail vein injection of adenovirus, respectively), andsubjected to HE staining and TUNEL staining.

As shown in FIG. 3, while in the Ad.dE1.3-administered group,hepatocytes exhibiting typical apoptotic morphology accompanyinginfiltration of neutrophils and macrophages, corresponding to theelevation in the level of liver enzyme (FIGS. 2B and 2C), were observedat 24 hr later, in the Ad.HB-EGF-administered group, as well as in theAd.HGF-administered group, apoptotic morphology of hepatocytes was notobserved. Furthermore, at 36 hr later, while in theAd.dE1.3-administered group, dropout of liver tissue due to progressionof hepatocellular apoptosis was observed, and also in theAd.HGF-administered group inflammation was observed, in the Ad.HB-EGF,disorder of liver tissue was almost completely inhibited.

Furthermore, TUNEL staining was performed in order to evaluatehepatocellular apoptosis in detail; as a result, both at 24 hr and 36 hrlater, compared to the number of TUNEL-positive hepatocytes in theAd.dE1.3-administered group, the positive cell number was significantlysmaller in the Ad.HB-EGF-administered group and Ad.HGF group, thereby itwas demonstrated that in Ad.HB-EGF- and Ad.HGF-administered group,hepatocellular apoptosis, which is an essence of fulminant hepaticfailure, was inhibited.

Experimental Example 4 Promoting Effect of Liver Regeneration byAd.HB-EGF

Liver regeneration, which is an index of essential therapeutic effect offulminant hepatic failure, was evaluated with the ratio of proliferatinghepatocytes identified by anti-Ki-67 mouse monoclonal antibody (clonename, TEC-3, Dako Cytomation, Denmark). Immunostaining with anti-Ki-67antibody was performed using paraffin sections of mouse liver at 24 hrand 36 hr after the agonist antibody administration (96 hr and 108 hrafter the tail vein injection of adenovirus, respectively).

As shown in FIG. 5, while in the Ad.dE1.3-administered group,Ki-67-positive cells, which signifies the growth of hepatocyte, werehardly detected both at 24 and 36 hr later, in theAd.HB-EGF-administered group, as well as in the Ad.HGF group,significant increase in Ki-67-positive cell was observed. Particularly,the ratio of Ki-67-positive cell at 24 hr later in theAd.HB-EGF-administered group was about 1.5-fold higher compared to HGF,only which has been reported as a potent therapeutic factor forfulminant hepatic failure, thereby it was demonstrated that HB-EGF hadtherapeutic effect for fulminant hepatic failure surpassing HGF.

These results suggest that HB-EGF, which is expressed in liver afteradministration of the Ad.HB-EGF, be effective in preventing onset andprogression of the pathology by strongly inhibiting hepatocellularapoptosis, which is an essence of fulminant hepatic failure, and ininducing an essential healing by promoting liver regeneration,specifically proliferation of surviving hepatocytes. Accordingly, thepharmaceutical agent of the present invention using HB-EGF can be alsoapplied to any other diseases accompanying liver damage (hepatocytedeath).

INDUSTRIAL APPLICABILITY

HB-EGF exhibits strong actions of inhibiting liver damage and apoptosisand inducing liver regeneration; therefore, HB-EGF or nucleic acidsencoding same are extremely useful as a drug for prevention andtreatment of various diseases accompanying liver damage or hepatocytedeath, particularly liver diseases such as fulminant hepatic failure.

While the present invention has been described with emphasis onpreferred embodiments, it is obvious to those skilled in the art thatthe preferred embodiments can be modified. The present invention intendsthat the present invention can be embodied by methods other than thosedescribed in detail in the present specification. Accordingly, thepresent invention encompasses all modifications encompassed in the gistand scope of the appended “CLAIMS.”

This application is based on patent application No. 2005-283085 filed inJapan, and the contents disclosed therein are hereby entirelyincorporated by reference. In addition, the contents disclosed in anypublication cited herein, including patents and patent applications, arehereby incorporated in their entireties by reference, to the extent thatthey have been disclosed herein.

1-12. (canceled)
 13. A method for protecting liver in a mammal, whichcomprises administering to said mammal an effective amount of aheparin-binding EGF-like growth factor or a partial peptide thereof. 14.The method of claim 13, which is also for promoting liver regeneration.15. The method of claim 13, which is used for the prophylaxis/treatmentof a liver disease.
 16. The method of claim 15, wherein the liverdisease is fulminant hepatic failure or acute hepatitis.
 17. The methodof claim 15, wherein the liver disease is selected from the groupconsisting of chronic hepatitis, autoimmune liver disease, viralhepatitis, liver fibrosis, cirrhosis, liver carcinoma, alcoholic liverdisease, drug-induced liver disease, liver abscess, hepatic parasitosis,hepatic amyloidosis and lupoid hepatitis.
 18. A method for promotingliver regeneration in a mammal with a liver disease or liver cell death,which comprises administering to said mammal an effective amount of aheparin-binding EGF-like growth factor or a partial peptide thereof. 19.The method of claim 18, which is used for the prophylaxis/treatment of aliver disease.
 20. The method of claim 19, wherein the liver disease isfulminant hepatic failure or acute hepatitis.
 21. The method of claim19, wherein the liver disease is selected from the group consisting ofchronic hepatitis, autoimmune liver disease, viral hepatitis, liverfibrosis, cirrhosis, liver carcinoma, alcoholic liver disease,drug-induced liver disease, liver abscess, hepatic parasitosis, hepaticamyloidosis and lupoid hepatitis.